DNA Manipulation (chapter 4) Flashcards
Polymerase Enzyme
An enzyme that builds a polymer
Synthesizes long chains of amino acids
DNA polymerase synthesizes DNA in a 5’ to 3’ ratio using an existing strand as a template
Ligase
Completes DNA strand by creating sugar-phosphate bonds between Okazaki fragments
Okazaki Fragment
‘Lagging Strand’ (5’) is replicated in short segments by DNA Polymerase
Endonuclease
Enzyme that cuts sugar-phosphate bonds between nucleotides
Palindrome
Sequence of double stranded DNA that reads the same on both strands in a 5’ to 3’ direction
Blunt Ends
If restriction site is cut directly down the middle
Sticky Ends
If cut with overhanging lengths of single stranded DNA, complemented by other ‘sticky ends’ from the same enzyme
Polymerase Chain Reaction
A laboratory technique used to produce many identical copies of DNA from a small initial sample (DNA Amplification)
PCR Process
- Identify 25 (or so) sequences at the 3’ end of each strand
- DNA heated to 95*c, where DNA denatures due to heat
- DNA primers are created, complementary to 25 bases, made using synthesizers
- Primers cool DNA to 55*c
- Reheated to 72c - Taq. polymerase extends primers, because 72 is optimal temperature
Primer
Short length of DNA complementary to 3’ end
Taq. Polymerase
DNA polymerase taken from bacteria Thermus Aquaticus
CRISPR
A naturally occuring adaptive immune response within prokaryotes
Adaptive Immune System
System can learn from infection and become more efficient
PAM
Protospacer Adjacent Motif (repeating pattern)
a sequence of two-six nucleotides that is found immediately next to the DNA targeted by Cas9
Spacer
DNA captured from bacteriophage viruses that have previously infected the cell
CAS 1 & 2
Separate enzymes, work in tandem, look for bases GG (guanine, guanine) and the base before that
GG Base
Recognised as PAM
crRNA
CRISPR RNA
gRNA
Guide RNA
Tracr RNA
trans-acting CRISPR RNA
CRISPR Sumamry
1- Bacteriophase inserts viral DNA into bactera cell
2- RNA polymerase creates guide RNA (gRNA)
- Transcription occurs
- CRISPR RNA formed
- Endonuclease cuts crRNA and transRNA into targetted gene sequence
3- CAS-9 attaches to gRNA
4- CAS-9 enzyme scanes DNA until it binds to PAM sight (…gg)
5- CAS-9 Binds to PAM site, begins unzipping complementary sequences
6- If gRNA is complementary to DNA, CAS-9 will cut DNA sequence nucleotides from PAM sequence
Anneal
Combines
CAS-9 Structure
Protein with a single polypeptide, but six domains
sgRNA
synthetic guide RNA
used to direct CAS-9 anywhere they want to cut
How CAS-9 cuts DNA
- After finding PAM, CAS separates strands, compares base sequences in spacer region of gRNA to bases upstream of PAM, on opposite strand
- If the sequence is complementary, DNA is cut 4 bases up from PAM
Non homologous end Joining
- Joins ends
- Error-prone, can cause mutation
Homology Directed Repair
Insert gene into genome to produce gene that wasnt possible before
Genome
complete set of DNA including genes within an organism
contains info to build and maintain genome
DNA technologies
Able to isolate and study fragments of DNA
Used to:
- understand evolution
- Investigate disease
- Develop products for medicine
Tools for studying and manipulating DNA
Naturally occuring enzymes usd by the cell for specific functions
Including:
- Endonucleases
- Ligases
- Polymerases
Plasmid
Clone short segments of Proteins
Gel Electrophoresis
A technique that separates DNA fragments based on their molecular size
Gel Electrophoresis Process - Preparing the Gel
- Special genetic grade agarose gel melted and poured
- When gel sets, it forms a pourous structure that DNA can move through - and other fluids
- Gek tray placed in electrophoresis chamber containing buffer
Buffer
clear solution, contains ions with electric current
Gel Electrophoresis Process - Loading Gel
- Gel (w/ DNA) pipetted into wells at negative electrode of electrophoresis tank
- Electrophoresis chamber connected to power supply with electrode near DNA
- Shows where DNA is
- DNA moves toward positive electrode
- Smaller strands are able to move further before becoming stuck in matrix
STR
Short randem respects (short sequence of DNA)
Recombinant Plasmids Process
1) Choose Restriction Enzyme
2) Choosing Plasmid
3) Using Restriction Endonuclease
4) Making a recombinant plasmid
5) Transforming Bacteria
6) Identifying and Culturing Bacteria
Recombinant Plasmid Process - Step 1) Choose Restriction Enzyme
Endonuclease is chosen to cut upstream and downstream of gene
Leaves sticky ends
(must cut palindromic region)
Recombinant Plasmid Process - Step 2) Choosing a Plasmid
Plasmid is chosen that has 2 genes that each encode observable traits
Of these, one must contain the restriction site
Recombinant Plasmid Process - Step 3) Using Restriction Endonuclease
Same restriction enzyme used to cut both source gene, and the plasmid
Recombinant Plasmid Process - Step 4) Making a Recombinant Plasmid
When source gene and plasmid are mixed, source gene may incorporate into plasmid, creating a recombinant
Plasmid
Extra-chromosomal loop of DNA
Recombinant
Organism or DNA molecule containing DNA from more than one species
Recombinant Plasmid Process - Step 5) Transforming Bacteria
Bacteria made competent to take up plasmid
- only some bacteria will take up plasmid
- only some will take up recombinant plasmid
Recombinant Plasmid Process - Step 6) Identifying and Culturing Bacteria
Growing bacteria on agar plate can help determine whether the bacteria has taken up a plasmid
Bacteria that haven’t taken a plasmid wont be able to grow as they lack resistance gene
Transgenic Organisms
An organism containing a gene from another species
Genetically Modified Organism
Any organism whose DNA has been modified by gene technology
RNAI technology
Technique where a gene is silenced by blocking its mRNA
Issues with GMO - Animals
- Animal genes in plant products
- Pig genes in other food - not koscher/halal
- Animal viruses may affect humans
Issues with GMO - Plants
- Concern of spread of GMO - cross pollination
- New allergens - soybean genes from nuts
Issues with GMO - Major Issues
- Monoculture - uniform selective, without variety
- Loss of genetic diversity - vulnerability
What does CRISPR Stand for
Clustered regularly interspaced short palindromic repeats
